Affinity chromatography of mammalian and yeast nucleosomes. Two modes of binding of transcriptionally active mammalian nucleosomes to organomercurial-agarose columns, and contrasting behavior of the active nucleosomes of yeast.

The reasons for the selective binding of nucleosomes from transcriptionally active genes to the organomercurial-agarose columns have been investigated. At least two modes of binding are identified by a new two-stage elution procedure that discriminates between nucleosomes which are retained by the Hg-column because of their salt-labile associations with SH-reactive non-histone proteins, and nucleosomes in which a conformational change has made the thiol groups of histone H3 accessible to SH-reagents. The first class is released from the column in 0.5 M NaCl; the second class is eluted in 10 mM dithiothreitol which displaces the bound H3-thiols. In mammalian cells, both classes of Hg-bound nucleosomes are enriched in the DNA sequences being transcribed at the time, and their histones H3 and H4 are hyperacetylated. In yeast cells, in which histone H3 lacks cysteinyl residues, only a small fraction of nucleosomes binds to the mercury column, and it has no enrichment of DNA sequences derived from the actively transcribed GAL, HIS4, and ACT1 genes. Since few nucleosomes remain on the column after elution in 0.5 M NaCl, the bound nucleosomes of yeast are retained primarily because of salt-labile associations with thiol-reactive nonhistone proteins. Thus, the presence of histone H3-thiol groups appears to be essential for the mercury binding of the second class of nucleosomes which, in mammalian cells, is derived from the transcriptionally active genes. The results support models of reversible nucleosome unfolding during transcription in mammalian cells to reveal previously inaccessible H3-SH groups, and they also indicate that other thiol-containing proteins, including high mobility group 1 and 2, become closely but transiently associated with the chromatin subunits during their transcription.